RAC1 inhibitors for the treatment of Alport glomerular disease

Abstract

The present invention provides methods of treating Alport syndrome in a subject by the administration of an agent that blocks the activation of RAC1/CDC42 members of the rho family of small GTPases. Such agents include, but are not limited to, the endothelin receptor antagonists such as bosentan and letairis and neutralizing antibodies to endothelin-1. Such administration prevents invasion of the glomerular capillary tufts by mesangial lamellipodial/filopodial processes, blocks mesangial process invasion, abrogates the deposition of laminin 211 in the GBM, and prevents the activation of maladaptive expression of proteins known to contribute to glomerular disease progression.

What is claimed is: 
     
       1. An in vitro method of identifying an endothelin receptor antagonist agent effective for inhibiting mesangial cell process invasion of the glomerular capillary loop in a kidney of a subject, the method comprising:
 contacting cultured mesangial cells with the agent; 
 contacting the cultured mesangial cells with endothelin-1; and 
 observing the formation of drebrin-positive filopodial microspikes in the mesangial cells; 
 wherein an effective agent inhibits the formation of drebrin-positive filopodial microspikes in the mesangial cells. 
 
     
     
       2. An in vitro method of identifying an endothelin receptor antagonist agent, the method comprising:
 contacting cultured mesangial cells with the agent; 
 contacting the cultured mesangial cells with endothelin-1; and 
 observing the formation of drebrin-positive filopodial microspikes in the mesangial cells; 
 wherein an effective agent inhibits the formation of drebrin-positive filopodial microspikes in the mesangial cells. 
 
     
     
       3. The method of  claim 2 , wherein the cultured mesangial cells comprise cultured primary mesangial cells. 
     
     
       4. The method of  claim 2 , wherein the cultured mesangial cells comprise murine cells. 
     
     
       5. The method of  claim 2 , wherein the formation of drebrin-positive filopodial microspikes is assayed by staining with an anti-debrin antibody. 
     
     
       6. The method of  claim 2 , wherein the formation of drebrin-positive filopodial microspikes is assayed by dual staining with phalloidin and an anti-debrin antibody. 
     
     
       7. The method of  claim 2 , wherein the agent comprises an antibody. 
     
     
       8. The method of  claim 2 , wherein the agent comprises an antibody that specifically binds endothelin-1. 
     
     
       9. The method of  claim 8 , wherein the antibody that specifically binds endothelin-1 neutralizes one or more functions of endothelin-1. 
     
     
       10. The method of  claim 2 , wherein the agent comprises an endothelin A receptor (ET A R) antagonist or a dual ET A R and ET B R antagonist. 
     
     
       11. The method of  claim 2 , further comprising assaying cell division cycle 42 (CDC42) protein activation, wherein an effective agent reduces CDC42 activation. 
     
     
       12. The method of  claim 11 , wherein assaying CDC42 activation comprises measuring GTP bound CDC42. 
     
     
       13. The method of  claim 12 , wherein GTP bound CDC42 is measured by ELISA. 
     
     
       14. The method of  claim 2 , further comprising:
 lysing the mesangial cells; and 
 assaying the cell lysate for activated cell division cycle 42 (CDC42) protein; 
 wherein assaying the cell lysate for activated CDC42 comprises measuring GTP bound CDC42; and 
 wherein an effective agent reduces the amount of GTP bound CDC42 in the cell lysate. 
 
     
     
       15. The method of  claim 2 , further comprising assaying the mesangial cells for perinuclear localization versus plasma membrane localization of cell division cycle 42 (CDC42) protein, wherein an effective agent demonstrates a relative absence in plasma membrane localization of CDC42. 
     
     
       16. The method of  claim 15 , wherein assaying the localization of CDC42 comprises immunofluorescent staining with an anti-CDC42 antibody. 
     
     
       17. The method of  claim 2 , wherein the agent inhibits endothelin-1 (ET-1) mediated endothelin A receptor (ET A R) activation and/or CDC42 activation. 
     
     
       18. The method of  claim 2 , wherein the agent inhibits CDC42 activation. 
     
     
       19. The method of  claim 2 , wherein the agent inhibits endothelin A receptor (ET A R)-mediated activation of CDC42 in mesangial cells.